A novel serine/threonine kinase associated with thrombin receptor signaling in human platelet

点击翻译按钮获取中文摘要

Novel protein kinases that may associate with thrombin receptor were sought by a procedure based on the ability of these enzymes to undergo renaturation and catalyze the phosphorylation of a protein substrate fixed in a gel. We employed a fusion protein comprising of glutathione S-transferase and the substrate protein that corresponds to residues 375-421 of thrombin receptor (GST-TRCD). We report that platelets contain a serine/threonine protein kinase with molecular masse of about 33 kDa, which specifically bound to GST-TRCD.Treatment of platelets with thrombin increased the thrombin receptor-associated kinase (TRAK) activity by 700%. TRAK was also activated by a thrombin receptor agonist peptide, but not by hirudin-treated or diisopropylphosphate-inactivated thrombin. Activation of thrombin receptor resulted in phosphorylation of TRCD by TRAK in a Ca^<2+>-dependent way. In summary, our results provide evidence of a novel ligand-activated serine/threonine kinase that associates with the cytoplasmic domain of the thrombin receptor.